Single Prolonged Stress Group Research Articles

Microbial composition, functionality, and stress resilience or susceptibility: unraveling sex-specific patterns

BackgroundFollowing exposure to traumatic stress, women are twice as likely as men to develop mood disorders. Yet, individual responses to such stress vary, with some people developing stress-induced psychopathologies while others exhibit resilience. The factors influencing sex-related disparities in affective disorders as well as variations in resilience remain unclear; however, emerging evidence suggests differences in the gut microbiota play a role. In this study, using the single prolonged stress (SPS) model of post-traumatic stress disorder, we investigated pre- and post-existing differences in microbial composition, functionality, and metabolites that affect stress susceptibility or resilience in each sex.MethodsMale and female Sprague–Dawley rats were randomly assigned to control or SPS groups. Two weeks following SPS, the animals were exposed to a battery of behavioral tests and decapitated a day later. Based on their anxiety index, they were further categorized as SPS-resilient (SPS-R) or SPS-susceptible (SPS-S). On the day of dissection, cecum, and selected brain tissues were isolated. Stool samples were collected before and after SPS, whereas urine samples were taken before and 30 min into the SPS.ResultsBefore SPS exposure, the sympathoadrenal axis exhibited alterations within male subgroups only. Expression of tight junction protein claudin-5 was lower in brain of SPS-S males, but higher in SPS-R females following SPS. Across the study, alpha diversity remained consistently lower in males compared to females. Beta diversity revealed distinct separations between male and female susceptible groups before SPS, with this separation becoming evident in the resilient groups following SPS. At the genus level, Lactobacillus, Lachnospiraceae_Incertae_Sedis, and Barnesiella exhibited sex-specific alterations, displaying opposing abundances in each sex. Additionally, sex-specific changes were observed in microbial predictive functionality and targeted functional modules both before and after SPS. Alterations in the microbial short-chain fatty acids (SCFAs), were also observed, with major and minor SCFAs being lower in SPS-susceptible males whereas branched-chain SCFAs being higher in SPS-susceptible females.ConclusionThis study highlights distinct pre- and post-trauma differences in microbial composition, functionality, and metabolites, associated with stress resilience in male and female rats. The findings underscore the importance of developing sex-specific therapeutic strategies to effectively address stress-related disorders.HighlightsSPS model induces divergent anxiety and social behavioral responses to traumatic stress in both male and female rodents.SPS-resilient females displayed less anxiety-like behavior and initiated more interactions towards a juvenile rat than SPS-resilient males.Sex-specific pre-existing and SPS-induced differences in the gut microbial composition and predictive functionality were observed in susceptible and resilient rats.SPS-resilient males displayed elevated cecal acetate levels, whereas SPS-susceptible females exhibited heightened branched-chain SCFAs.

Maternal deprivation causes CaMKII downregulation and modulates glutamate, norepinephrine and serotonin in limbic brain areas in a rat model of single prolonged stress

BackgroundEarly life stress is a major risk factor for later development of psychiatric disorders, including post-traumatic stress disorder (PTSD). An intricate relationship exists between various neurotransmitters (such as glutamate, norepinephrine or serotonin), calcium/calmodulin-dependent protein kinase II (CaMKII), as an important regulator of glutamatergic synaptic function, and PTSD. Here, we developed a double-hit model to investigate the interaction of maternal deprivation (MD) as an early life stress model and single prolonged stress (SPS) as a PTSD model at the behavioral and molecular levels. MethodsMale Wistar rats exposed to these stress paradigms were subjected to a comprehensive behavioral analysis. In hippocampal synaptosomes we investigated neurotransmitter release and glutamate concentration. The expression of CaMKII and the content of monoamines were determined in selected brain regions. Brain-derived neurotrophic factor (BDNF) mRNA was quantified by radioactive in situ hybridization. ResultsWe report a distinct behavioral phenotype in the double-hit group. Double-hit and SPS groups had decreased hippocampal presynaptic glutamatergic function. In hippocampus, double-hit stress caused a decrease in autophosphorylation of CaMKII. In prefrontal cortex, both SPS and double-hit stress had a similar effect on CaMKII autophosphorylation. Double-hit stress, rather than SPS, affected the norepinephrine and serotonin levels in prefrontal cortex, and suppressed BDNF gene expression in prefrontal cortex and hippocampus. LimitationsThe study was conducted in male rats only. The affected brain regions cannot be restricted to hippocampus, prefrontal cortex and amygdala. ConclusionDouble-hit stress caused more pronounced and distinct behavioral, molecular and functional changes, compared to MD or SPS alone.

Intra-mPFC injection of sodium butyrate promotes BDNF expression and ameliorates extinction recall impairment in an experimental paradigm of post-traumatic stress disorder.

Objective(s):Therapeutic strategies that facilitate extinction are promising in the treatment of post-traumatic stress disorder (PTSD). Brain-derived neurotrophic factor (BDNF) has a crucial role in neural plasticity, a process needed for the retention of fear extinction. In this study, we investigated the effects of local administration of a histone deacetylase (HDAC) inhibitor, sodium butyrate (NaBu), on BDNF transcription and behavioral markers of extinction in the single prolonged stress (SPS) model of PTSD. Materials and Methods:NaBu was infused into the infralimbic (IL) subregion of the medial prefrontal cortex (mPFC) of male rats. The freezing response was recorded as the criterion to assess fear strength on the day of extinction as well as 24 hr later in the retention test. Other behavioral tests were also measured to evaluate the anxiety level, locomotor activity, and working memory on the retention day. HDAC activity and BDNF mRNA expression were evaluated after the behavioral experiments. Results: NaBu facilitated the recall of fear extinction in SPS rats (P<0.0001). SPS rats had higher HDAC activity (P<0.0001) and lower BDNF expression (P<0.05) than non-SPS animals. Also, anxiety was higher in the SPS group (P<0.0001), but locomotor activity (P=0.61) and working memory (P=0.36) were not different between SPS and Non-SPS groups.Conclusion:Our findings provide evidence that the mechanism of action of NaBu in the improvement of extinction recall is mediated, in part, by enhancing histone acetylation and reviving BDNF expression in IL.

Resilience or susceptibility to traumatic stress: Potential influence of the microbiome

Exposure to traumatic stress is a major risk factor for development of neuropsychiatric disorders in a sub-population of individuals, while others remain resilient. The mechanisms and contributing factors differentiating between these phenotypes are still unclear. We hypothesize that inter-individual differences in the microbial composition and function contribute to host resilience or susceptibility to stress-induced psychopathologies. The current study aimed to characterize gut microbial community before and after exposure to traumatic stress in an animal model of PTSD. Sprague-Dawley male rats were randomly divided into unstressed controls and experimental group subjected to Single Prolonged Stress (SPS). After 14 days, behavioral analyses were performed using Open Field, Social Interaction and Elevated Plus Maze tests. Based on the anxiety measures, the SPS group was further subdivided into resilient (SPS-R) and susceptible (SPS–S) cohorts. The animals were sacrificed after the last behavioral test and cecum, colon, hippocampus, and medial prefrontal cortex were dissected. Prior to SPS and immediately after Open Field test, fecal samples were collected from each rat for 16S V3–V4 ribosomal DNA sequencing, whereas urine samples were collected before SPS, 90 min into immobilization and on the day of sacrifice to measure epinephrine and norepinephrine levels. Analyses of the fecal microbiota revealed significant differences in microbial communities and in their predictive functionality among the groups before and after SPS stressors. Before SPS, the SPS-S subgroup harbored microbiota with an overall pro-inflammatory phenotype, whereas SPS-R subgroup had microbiota with an overall anti-inflammatory phenotype, with predictive functional pathways enriched in carbohydrate and lipid metabolism and decreased in amino acid metabolism and neurodegenerative diseases. After SPS, the gut microbial communities and their predictive functionality shifted especially in SPS cohorts, with volatility at the genus level correlating inversely with Anxiety Index. In line with the alterations seen in the gut microbiota, the levels of cecal short chain fatty acids were also altered, with SPS-S subgroup having significantly lower levels of acetate, valerate and caproate. The levels of acetate inversely correlated with Anxiety Index. Interestingly, urinary epinephrine and norepinephrine levels were also higher in the SPS-S subgroup at baseline and during stress, indicative of an altered sympathoadrenal stress axis. Finally, shorter colon (marker of intestinal inflammation) and a lower claudin-5 protein expression (marker for increased blood brain barrier permeability) were observed in the SPS-S subgroup. Taken together, our results suggest microbiota is a potential factor in predisposing subjects either to stress susceptibility or resilience. Moreover, SPS triggered significant shifts in the gut microbiota, their metabolites and brain permeability. These findings could lead to new therapeutic directions for PTSD possibly through the controlled manipulation of gut microbiota. It may enable early identification of individuals more likely to develop prolonged anxiogenic symptoms following traumatic stress.

Single Prolonged Stress Alters Vasopressin and Orexin System Expression in Sprague Dawley Rats

Posttraumatic stress disorder (PTSD) is a complex psychiatric disorder triggered by a terrifying event, causing severe anxiety and nightmares. Studies showed that patients with PTSD have increased risk of cardiovascular disease. However, the effect of PTSD on blood pressure remains unraveled. In this study, we test the hypothesis that PTSD alters expression of arginine vasopressin (AVP) and orexin system components and therefore increases blood pressure. Single prolonged stress (SPS) protocol including 2 hours of restraint, a 20‐min forced swimming and exposure to diethyl ether until loss of consciousness was used to create a PTSD model. Eight‐week‐old male Sprague Dawley (SD) rats were divided into two groups and treated with or without SPS, then all rats were single‐housed and remained undisturbed for 7 days. Their blood pressure (BP) and heart rates (HR) were recorded by radio telemetry transmitter system (Data Science International) prior to the treatment as baseline, and 1‐ day, and 7‐day post the treatment. The data showed that SPS significantly increased BP (ΔBP: 8.384 ± 1.187 mmHg, N=6 P&lt;0.05) and HR (ΔHR: 32.88 ± 9.149 beats/min, N=6 P&lt;0.05) compared to control rats (ΔBP: 2.346 ± 0.9324 mmHg, ΔHR: ‐1.503 ± 5.960 beats/min, N=8) on day 1 post treatment, but no significance difference was observed in BP and HR on day 7 compared to their own baseline and between SPS and control groups.To investigate whether AVP and orexin systems participate in PTSD development, we examined paraventricular nucleus (PVN) and adrenal gland AVP‐ and orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) expression in both SD male and female rats with or without SPS at day 7. Our data showed that PVN AVP mRNA levels were increased by 2‐fold in the SPS group compared to control in male rats (P&lt;0.05) but this change was not observed in female rats. No changes were observed in PVN OX1R and OX2R mRNA levels between SPS and control rats in both males and females. Peripheral orexin receptors’ protein levels were altered but showed inconsistency in male and female rats: western blot data showed that adrenal gland OX1R protein levels were increased in the SPS group (control: 1.000 ± 0.07424 N=5 vs SPS: 1.531 ± 0.2016 N=5; P&lt;0.05) in male rats, while OX2R protein levels were decreased in female rats in female rats (control: 1.000 ± 0.01025 N=5 vs SPS: 0.8840 ± 0.04533 N=6; P&lt;0.05). Renal OX1R was also increased (control: 1.000 ± 0.02794 N=3 vs SPS: 1.374 ± 0.1385 N=3; P&lt;0.06) in female rats under SPS. ELISA assay showed that plasma orexin A levels were increased in female rats under SPS (control: 1.000 ± 0.1600 pg/mL N=3 vs SPS: 1.561 ± 0.1262 pg/mL; N=3 P&lt;0.05) but not in male rats.These results suggested that the central AVP system and peripheral orexin system may play a role in PTSD‐related high blood pressure development, and the peripheral orexin system may mediate stress response of female rats more than male rats. These molecular differences between SD male and female rats may also help to answer why females and males showed different responses to trauma. Further study will continue to find the PTSD‐sensitized hypertensive model and investigate specific functions of AVP and orexin system in PTSD.

Alterations in Microbiota‐Gut‐Brain Axis and Susceptibility or Resilience to Traumatic Stress

Post‐Traumatic Stress Disorder (PTSD) is a severe and long‐lasting outcome of exposure to traumatic life event. Exposure to trauma induces PTSD only in a sub‐population of individuals while others remain resilient. The mechanisms and contributing factors differentiating between stress resilient and stress susceptible subjects are still unclear. We hypothesize that inter‐individual differences in the microbiome composition and its function contribute to host resilience or susceptibility to stress induced psychopathologies. The current study aimed to characterize differences in the gut microbial community structure and function that mediate vulnerability or resilience to traumatic stress in the Single Prolonged Stress (SPS) animal model of PTSD. Sprague‐Dawley male rats were divided into unstressed controls and SPS experimental group. After 14 days, behavioral analyses were performed using Open Field, Social Interaction and Elevated Plus Maze tests. Based on the anxiety measures, the SPS group was further subdivided into SPS‐resilient (R) and SPS‐susceptible (S) subgroups. After the last behavioral test, the animals were sacrificed and cecum, colon, hippocampus, and medial prefrontal cortex were collected. Prior to SPS and immediately after Open Field test, fecal samples were collected from each rat for 16S V3‐V4 ribosomal sequencing; whereas urine samples were collected before SPS, 90 minutes into immobilization and on the day of dissection to measure urinary epinephrine (epi) and norepinephrine (norepi) levels. Analyses of the fecal microbiota revealed significant differences among the groups before and after SPS stressors. Before SPS, the SPS‐S subgroup harbored microbiota with an overall pro‐inflammatory phenotype, such as significantly increased levels of Clostridia, and decreased levels of Lactobacillusand Barnesiella compared to the SPS‐R subgroup. After SPS, among the many changes observed were significant shifts in the abundance of Firmicutes and Bacteroidetes in SPS subgroups. Urinary epi and norepi levels were also different among the groups, with SPS‐S subgroup having higher baseline catecholamines levels. In line with the alterations seen in the gut microbiota, the levels of cecal short chain fatty acids were also altered, with SPS‐S subgroup having significantly lower levels of Acetate, Valerate and Caproate. Interestingly, the levels of acetate inversely correlated with anxiety index. The colon length and permeability were also altered, with SPS‐S having significantly shorter colon and increased permeability as measured by Claudin‐1 expression. Finally, analyses of different brain regions also revealed significant increases in blood brain barrier permeability in SPS‐S subgroup as determined by Claudin‐5 expression. Taken together, our results suggest microbiota is a potential factor in predisposing subjects either to stress susceptibility or resilience. Moreover, SPS triggered significant shifts in the gut microbiota, their metabolites and brain and gut permeability. These findings could lead to new therapeutic directions for PTSD possibly through the controlled manipulation of gut microbiota. It may enable early identification of individuals more likely to develop prolonged anxiolytic symptoms following traumatic stress.

An Advanced Transcriptional Response to Corticosterone After Single Prolonged Stress in Male Rats.

Stress-related neuropsychiatric disorders are often accompanied by dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis. In patients suffering from post-traumatic stress disorder (PTSD), increased sensitivity of glucocorticoid negative feedback has regularly been observed. The single prolonged stress (SPS) paradigm was developed to model increased negative feedback and other aspects of PTSD in rats. In this study, we used a setup that precluded the evaluation of negative feedback but rather served to test the hypothesis of the enhanced glucocorticoid receptor (GR) signaling in higher brain areas. We injected corticosterone or vehicle 7 days after SPS and evaluated plasma corticosterone, as well as gene expression in the dorsal hippocampus and amygdala. We observed a strikingly rapid change in the expression of established GR target genes (t = 30 min) only in the SPS group on exogenous corticosterone injection. Our results extend the notion of increased GR sensitivity in PTSD to include transcriptional responses in the hippocampus.

A prognostic study on the effect of post-traumatic stress disorder on cerebral ischaemia reperfusion-induced stroke

Objectives Previous studies have been established that persons who experienced a stroke are soon likely to develop several anxiety disorders. In which one of the major anxiety disorders is Post-traumatic Stress Disorder (PTSD). Yet, the likelihood of PTSD in conjunction with cerebral stroke has not been well described. Hence, we evaluated the impact of PTSD on cerebral stroke in rodents subjected to single prolonged stress (SPS) and bilateral common carotid artery occlusion (BCCAo), respectively. Methods The relation between PTSD and cerebral stroke is evaluated by performing behavioural, biochemical, histopathological, and brain lesion area measurement studies. Results Interestingly, SPS + BCCAo induction increased behavioural abnormalities like cognitive impairment and anxiety-like behaviour compared to SPS and BCCAo groups alone. Motor impairment was also observed in SPS + BCCAo rats compared to SPS rats, whereas no change with BCCAo rats. Furthermore, increased brain tissue MDA, acetylcholinesterase, and decreased SOD, catalase, and GSH were observed in SPS + BCCAo subjected rats compared to SPS and BCCAo rats alone. Additionally, SPS + BCCAo induction considerably increased the plasma corticosterone levels and caused severe neurotransmitter alterations. The SPS + BCCAo exposure significantly increased the brain lesion area in comparison with BCCAo rats. Moreover, severe histopathological alterations were observed in the hippocampus (CA1) of SPS + BCCAo rats compared to SPS and BCCAo rats alone. Conclusions In conclusion, our study results suggested that SPS-induced PTSD may aggravate the BCCAo induced cerebral ischaemia-reperfusion injury.

What Happened in the Hippocampal Axon in a Rat Model of Posttraumatic Stress Disorder.

Studies from postmortem and animal models have revealed altered synapse morphology and function in the brain of posttraumatic stress disorder (PTSD). And the effects of PTSD on dendrites and spines have been reported, however, the effection on axon include microtubule (MT) and synaptic vesicles of presynaptic elements remains unknown. Hippocampus is involved in abnormal memory in PTSD. In the present study, we used the single prolonged stress (SPS) model to mimic PTSD. Quantitative real-time polymerase chain reaction (RT-qPCR) and high-throughput sequencing (GSE153081) were utilized to analyze differentially expressed genes (DEGs) in the hippocampus of control and SPS rats. Immunofluorescence and western blotting were performed to examine change in axon-related proteins. Synaptic function was evaluated by measuring miniature excitatory postsynaptic currents (mEPSCs). RNA-sequencing analysis revealed 230 significantly DEGs between the control and SPS groups. Gene Ontology analysis revealed upregulation in axonemal assembly, MT formation, or movement, but downregulation in axon initial segment and synaptic vesicles fusion in the hippocampus of SPS rats. Increased expression in tau, β-tubulin MAP1B, KIF9, CCDC40, DNAH12 and decreased expression in p-tau, stathmin suggested SPS induced axon extension. Increased protein expression in VAMP, STX1A, Munc18-1 and decreased expression in synaptotagmin-1 suggested SPS induced more SNARE complex formation but decreased ability in synaptic vesicle fusion to presynaptic active zone membrane in the hippocampus of SPS rats. Further, low mEPSC frequency in SPS rats indicated dysfunction in presynaptic membrane. These results suggest that axon extension and synaptic vesicles fusion abnormality are involved in dysfunction of PTSD.

Activation of HDAC4 and GR signaling contributes to stress-induced hyperalgesia in the medial prefrontal cortex of rats

“Stress-induced hyperalgesia (SIH)” is a phenomenon that stress can lead to an increase in pain sensitivity. Epigenetic mechanisms have been known to play fundamental roles in stress and pain. Histone acetylation is an epigenetic feature that is changed in numerous stress-related disease situations. However, epigenetic mechanism for SIH is not well known. We investigated the effect of histone acetylation on pain hypersensitivity using SPS (single-prolonged stress) + CFA (complete Freund’s adjuvant) model. We showed that the glucocorticoid receptor (GR)-pERK-pCREB-Fos signaling pathway was upregulated on stress-induced hyperalgesia and the paw withdrawal threshold in the SPS + CFA group dropped significantly compared with the SPS or CFA group. Histone deacetylases 4 (HDAC4)-expressing neurons in the medial prefrontal cortex (mPFC) were increased in the SPS + CFA-exposed group compared with CFA-exposed or SPS-exposed group. And we showed that the effects of stress-induced hyperalgesia were critically regulated via reversible acetylation (HDAC4) of the GR. Inhibiting HDAC4 by microinjection of sodium butyrate into the mPFC could disrupt glucocorticoid receptor (GR) signaling pathway, which lowered SPS + CFA-caused mechanical allodynia and alleviated anxiety-like behavior. Together, our studies suggest that HDAC inhibitors might involve in the process of stress-induced hyperalgesia.

Histological study on the effect of single prolonged stress model on the hippocampus of adult male albino rat

Abstract Introduction: Post- traumatic stress disorder (PTSD) is a psychiatric disorder which occurs after the experience of life threatening events as natural disasters, military combat, serious accidents, or violent sexual assaults. It has a severe impact on quality of life. Aim of the work to study the effect of single prolonged stress on the structure of hippocampus in adult male albino rats. Materials and Methods 20 adult male albino rats were randomly divided into four groups control group (group I) and single prolonged stress group examined at day 1(group II), day 7 (group III) and day 14 (groupIV). Rats were restrained by placing them in plastic restrainers, immediately followed by 20 minutes of forced swimming in 20–24°C water. Rats were exposed to ether until general anesthesia then they were placed in their home cages. At the end of the experiment, the hippocampi were taken and processed for light microscopic study. Results pyramidal cells of the CA3 region of the hippocampus showed progressive degeneration with passage of time from day 1 to day 14 with decrease in thickness. Transient increase in glial fibrillary acidic protein (GFAP) expression in the astrocytes of the hippocampus was detected at day 1 followed by reduction in GFAP expression started at day seven and continued to day 14. Conclusion post-traumatic stress PTSD causes significant structural hippocampal neuronal damage affecting the CA3 hippocampal subfield.

D-Cycloserine Facilitates Reversal in an Animal Model of Post-traumatic Stress Disorder

Many psychiatric disorders are associated with cognitive dysfunction that is ineffectively treated by existing pharmacotherapies and which may contribute to poor real-world functioning. D-cycloserine (DCS) is a partial N-methyl-D-aspartate (NMDA) agonist that has attracted attention because of its cognitive enhancing properties, including in people with post-traumatic stress disorder (PTSD). Here, we examined the effect of DCS on reversal learning - a type of cognitive flexibility – following exposure to single prolonged stress (SPS), a rodent model of PTSD. Male Sprague Dawley rats (n = 64) were trained to press levers in an operant chamber, matched for performance and assigned to SPS or control (unstressed) groups. Following SPS, rats received three additional lever press sessions, followed by a side bias test on day three. One day later they learned a response discrimination rule (press left or right lever, opposite to side bias) and on a subsequent day were trained (and tested) for reversal to the opposite lever. DCS (15 mg/kg) or vehicle was administered 30 minutes prior to the reversal session. No between-group differences were found in acquisition or retrieval of the initial rule, but a significant drug x stress interaction on response discrimination reversal indicated that DCS had a greater beneficial effect on SPS rats’ cognitive flexibility than it did on performance in controls. These findings add to a growing literature on the beneficial effects of DCS for treating a wide variety of deficits that develop following exposure to extreme stress and may have implications for the development of novel pharmacotherapies for PTSD.

Dexmedetomidine Alleviates Presurgical Anxiety-Induced Persistent Postsurgical Pain via Decreasing the Expression of Glucocorticoid Receptor

Background: Preoperative anxiety can worsen postsurgical pain. Glucocorticoids play an important role in psychological anxiety and pain, but the effect of glucocorticoid receptor (GR) on persurgical anxiety-induced persistent postsurgical pain remains unknown.Methods: Adult male Sprague Dawley rats were randomly divided into 10 groups: control group, SPS group, incision group, 'SPS-plus-incision' group, saline group, metyrapone group, Dexmedetomidine group 1 (10 μg/kg), Dexmedetomidine group 2 (20 μg/kg), Dexmedetomidine group 3 (40 μg/kg) and Dexmedetomidine + Corticosterone group. Single-prolonged stress (SPS) was used to induce anxiety behaviors. Intraperitoneal injection of saline and dexmedetomidine was performed at 24 h after SPS and 0.5 h before incision. Intraperitoneal injection of metyrapone (25 mg/kg) was performed at 1h before SPS. Paw withdrawal mechanical threshold (PWMT) was tested at 24 h before SPS and on 1, 4, 7, 14, 21, and 28 days after incision. Corticosterone levels were determined using ELISA. The expression of GR was determined using Western blot.Results: The 'SPS-plus-incision' group decreased PWMT compared with control group and incision group from 1 to 28 days (P<0.05). SPS combined with incision increased plasma corticosterone levels compared with control group (P<0.05). A time-dependent increase in GR was also observed in 'SPS-plus-incision' group (P<0.05). Metyrapone significantly blunted the SPS-induced persistent postsurgical pain (P<0.05). Intraperitoneal administration of dexmedetomidine inhibits SPS-induced persistent pain compared with group saline (P<0.05). The expression of GR decreased after the intraperitoneal administration of dexmedetomidine (P<0.05). Pretreatment with corticosterone blocked this effect.Conclusions: Glucocorticoids contributed to presurgical anxiety-induced persistent postsurgical pain. Dexmedetomidine that decreased the expression of GR alleviated anxiety-induced persistent pain. These results indicated that dexmedetomidine may be an effective agent for preventing presurgical anxiety-induced persistent postoperative pain. Citation: Lu Li, Zuo-Xia Zhang, Zhi-Yu Yin, Cui-E Lu, Yi-Shan Lei, Rao Sun, et al. Dexmedetomidine alleviates presurgical anxiety-induced persistent postsurgical pain via decreasing the expression of glucocorticoid receptor. J Anesth Perioper Med 2017; x: x-x. doi:10.24015/JAPM.2017.0013This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Alterations in cyclinD1 and cyclin‑dependent kinase 4 expression in the amygdalae of post‑traumatic stress disorder rats.

The amygdalae are an important component of the human limbic system and exhibit a key role in emotional and behavioral reactions. Previous studies have demonstrated abnormal function and morphology in the amygdalae of post‑traumatic stress disorder (PTSD)‑like animal models, however the underlying molecular mechanisms remain elusive. The authors have previously demonstrated that PTSD induced increased apoptosis in the amygdala of PTSD‑like animals. CyclinD1 and cyclin‑dependent kinase4 (CDK4) are two important regulators of the cell cycle. The study explored the expression of cyclinD1 and CDK4 in the amygdala in PTSD. The single‑prolonged stress (SPS) rat model was used as a PTSD‑like model. Ultrastructural alterations of cells in the amygdala were observed using transmission electron microscopy (TEM). 4',6‑Diamidino‑2‑phenylindole (DAPI) fluorescence was employed to detect nuclear pycnosis. CyclinD1 and CDK4 expression in the amygdala cells was examined using immunofluorescence, Western blotting and reverse transcription‑quantitative polymerase chain reaction. TEM revealed morphological alterations to the amygdala cells of the SPS rats. DAPI‑stained nuclear brightness levels differed between the control and SPS groups. Expression of cyclinD1 and CDK4 in the amygdala increased gradually 1day and 4days following SPS stimulation, and peaked 7days following SPS stimulation at the protein and mRNA levels, in comparison with the control rats. These findings suggest that SPS resulted in increased cyclinD1 and CDK4 expression, which may accelerate cell apoptosis. This may be associated with SPS‑induced abnormal function and structure of the amygdala.

Decreased Glutamatergic Activity in the Frontal Cortex of Single Prolonged Stress Model: In vivo and Ex Vivo Proton MR Spectroscopy.

Single prolonged stress (SPS) is one of the preclinical models of posttraumatic stress disorder (PTSD) in humans. Not every traumatized person develops PTSD and the onset of the disease varies from months to many years after exposure to life-threatening events. The pathogenetic neurometabolites in PTSD have not been investigated to date, and could provide a means for therapeutic interventions. Therefore the present study aimed to evaluate neurochemical changes in the frontal cortex in the SPS model during time-dependent sensitization using in vivo and ex vivo proton magnetic spectroscopy (1H-MRS). Twenty-one male Sprague-Dawley rats (200-220g) were randomly assigned into two groups (Control, n = 10; SPS, n = 11). SPS consists of three consecutive stressors (restraint, forced swimming, and ether exposure) followed by 7 days without disturbance. In vivo 1H-MRS scans were conducted at baseline, immediately after SPS, and 3 and 7 days after SPS to quantify time-dependent alterations in the frontal cortex. On day 7, all animals were sacrificed and ex vivo 1H-MRS was performed. After SPS exposure, the SPS group showed signs of excitatory activities (glutamate) and cellular membrane turnover (choline and total choline) for 7 days. After the time-sensitization period, the SPS group showed lower glutamate and creatine levels and higher choline and lactate levels than the control group. These results indicate that SPS induces sustained adaptation of glutamatergic neuronal activity in the frontal cortex. Therefore, we conclude that SPS-induced stress reduces glutamatergic metabolism in the frontal cortex.

Single-Prolonged-Stress-Induced Changes in Autophagy-Related Proteins Beclin-1, LC3, and p62 in the Medial Prefrontal Cortex of Rats with Post-traumatic Stress Disorder

Autophagy, or type II programmed cell death, plays a crucial role in many nervous system diseases. However, few studies have examined the role of autophagy in post-traumatic stress disorder (PTSD), and the mechanisms underlying PTSD are poorly understood. The objective of this research was to explore the expression of three important autophagy-related proteins, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), and p62/SQSTM1 (p62), in the medial prefrontal cortex (mPFC) of an animal model of PTSD to identify changes in autophagic activity during PTSD pathogenesis. PTSD was induced in rats by exposure to a single-prolonged stress (SPS). The Morris water maze was used to assess cognitive changes in rats from the SPS and control groups. Transmission electron microscopy (TEM) was employed to observe mPFC morphological changes. Immunohistochemistry, immunofluorescence, and Western blotting techniques were used to detect expression of Beclin-1, LC3, and p62 in the mPFC. The Morris water maze test results showed that the escape latency time was increased and that the percent time in the target quadrant was decreased in the SPS group compared with that in the control group. Numerous visible autolysosomes in mPFC neurons were observed using TEM after SPS stimulation. Compared with that in the control group, the expression of Beclin-1 and the LC3-II/I ratio significantly decreased at 1day, then increased and peaked at 7days, and slightly decreased at 14days after SPS stimulation, whereas the converse was found for p62 expression. In conclusion, dysregulation of autophagic activity in the mPFC may play a crucial role in PTSD pathogenesis.

Molecular Mechanisms of IRE1α-ASK1 Pathway Reactions to Unfolded Protein Response in DRN Neurons of Post-Traumatic Stress Disorder Rats.

The goal of this study was to further elucidate the molecular mechanisms of post-traumatic stress disorder (PTSD) pathogenesis and to provide experimental evidence for new drug targets for effective PTSD treatment. Expression changes of IRE1α, ASK1, and other downstream molecules of the IRE1α-ASK1 endoplasmic reticulum stress (ERS) signaling pathway were investigated. JNK, P38, CHOP, Bcl-2, and Bax were analyzed at both protein and mRNA levels of dorsal raphe nucleus (DRN) neurons of PTSD rats. The rat PTSD model was established via the single-prolonged stress (SPS) method. Animals were randomly divided into five groups: a normal control group, a 1-day SPS group, a 4-days SPS group, a 7-day SPS group, and a 14-day SPS group. Spatial memory and learning ability of rats were evaluated subsequent to SPS using the Morris water maze test. Changes of IRE1α expression in the control and SPS groups were detected via immunohistochemistry (IHC). Protein and mRNA expressions of IRE1α, ASK1, JNK, P38, CHOP, Bcl-2, and Bax in the control and SPS groups were detected via Western blot and RT-PCR, respectively. The Morris water maze test revealed significantly longer average escape latencies in all SPS groups compared to the control group. In the spatial probe test, the percentage of time spent in the target quadrant was significantly lower in the SPS groups compared to control. IHC revealed increased positive expression of IRE1α subsequent to SPS challenge, reaching maximal levels on days four and seven (P<0.01), while significantly decreasing on day 14 (P<0.01). Western blot and RT-PCR revealed that protein and mRNA expressions of IRE1α, ASK1, JNK, CHOP, and P38 were significantly increased compared to control, peaking on days one, four, and seven post-SPS before returning to previous levels. Compared to control, expressions of Bcl-2 and Bax presented an initial increasing tendency followed by a decrease. A peak of Bcl-2 expression appeared early on day one following SPS, then decreased to a steady level. Bax expression in the SPS groups remained constant during early stages after SPS (days one to three) compared to control; however, expression significantly increased on day four and maintained a high level. In summary, 1) SPS challenge significantly activated the IRE1α-ASK1-JNK and IRE1α-ASK1-P38 apoptosis-signaling pathways in DRN neurons of PTSD rats. This resulted in a cascade of downstream reactions and ultimately apoptosis of DRN neurons. 2) Increased expression of apoptosis-associated molecules Bcl-2 and Bax in DRN neurons following SPS challenge was revealed as a central mechanism, inducing apoptosis of DRN neurons in PTSD rats.

Iron-induced neuronal damage in a rat model of post-traumatic stress disorder

Previous studies have shown that iron redistribution and deposition in the brain occurs in some neurodegenerative diseases, and oxidative damage due to abnormal iron level is a primary cause of neuronal death. In the present study, we used the single prolonged stress (SPS) model to mimic post-traumatic stress disorder (PTSD), and examined whether iron was involved in the progression of PTSD. The anxiety-like behaviors of the SPS group were assessed by the elevated plus maze (EPM) and open field tests, and iron levels were measured by inductively coupled plasma optical emission spectrometer (ICP-OES). Expression of glucocorticoid receptors and transferrin receptor 1 (TfR1) and ferritin (Fn) was detected by Western blot and immunohistochemistry in selected brain areas; TfR1 and Fn mRNA expression were detected by quantitative-polymerase chain reaction (Q-PCR). Ultrastructures of the hippocampus were observed under a transmission electron microscope. Our results showed that SPS exposure induced anxiety-like symptoms and increased the level of serum cortisol and the concentration of iron in key brain areas such as the hippocampus, prefrontal cortex, and striatum. The stress induced region-specific changes in both protein and mRNA levels of TfR1 and Fn. Moreover, swelling mitochondria and cell apoptosis were observed in neurons in brain regions with iron accumulation. We concluded that SPS stress increased iron in some cognition-related brain regions and subsequently cause neuronal injury, indicating that the iron may function in the pathology of PTSD.

Stress-Induced Increases in Levels of Caspases in the Prefrontal Cortex in a Rat Model of PTSD

We investigated the levels of expression of caspase-3 and 9 in the medial prefrontal cortex (mPFC) of rats subjected to single prolonged stress (SPS) to provide a novel insight into the mechanism of how this cortical region is related to post-traumatic stress disorder (PTSD). Fifty male Wistar rats were divided into the control group and four SPS groups examined at days 1, 4, 7, and 14 after treatment. Expression of caspase-3 in SPS groups was significantly greater when compared with the control group (P < 0.05) and peaked at day 7 after exposure to SPS. In the control group, the intensity of fluorescence of caspase-9-positive cells was low, while that in the SPS groups was significantly higher (P < 0.01) and peaked at day 4 after exposure to SPS. After SPS episodes, levels of mRNA of caspase-3 and caspase-9, compared with those in the control group, gradually increased and peaked at days 7 and 4, respectively (P < 0.01). Therefore, changes of expression of caspase-9 and caspase-3 may play an important role in the pathogenesis of PTSD.

The Use of Thalidomide in Treating PTSD and Co‐morbid Pain in an Animal Model of PTSD

Single‐prolonged stress (SPS) is an established animal model for post‐traumatic stress disorder (PTSD). We have previously reported that SPS induces increases in serum TNF‐α levels as well as long‐lasting mechanical allodynia and thermal hyperalgesia at day 3 post SPS. The aim of this study was to determine the effects of TNF‐α blockade on development of PTSD and co‐morbid pain symptoms. To achieve this, male Sprague‐Dawley rats were subjected to SPS; half of the SPS and control group rats were immediately presented with 50 mg/kg thalidomide (THL), a potent TNF‐α blocker, in 10% DMSO vehicle, daily, i.p for 5 days; non‐SPS rats received vehicle injections. THL almost completely prevented mechanical allodynia and reduces thermal hyperalgesia associated with SPS (p&lt;0.05) that is normally noted as early as day 3 post SPS, without altering baseline sensitivity itself. Using a 2‐way ANOVA, a significant interaction was observed between THL and SPS (p&lt;0.0001). Additionally, utilizing the elevated plus maze test, THL treatment reduces anxiety like behaviors, specifically mobility time, immobility time, open entries and time spent in open arms (p&lt;0.05). Also, using a 2‐way ANOVA, a significant interaction was observed between THL and SPS (p&lt;0.05) This suggests that immunomodulatory drugs such as THL, may offer a novel therapeutic method to prevent or reduce symptoms of PTSD and co‐morbid pain if administered shortly after the traumatic event.SupportDepartment of the Army DMRDP W81XWH‐11‐2‐0077